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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
1986
Reference Type:
publication
Title:
Unnamed
Year:
2000

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
not specified
Remarks:
This information was not provided
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
Histidine-requiring gene in Salmonella typhimurium
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction from Aroclor 1254-induced male Sprague-Dawley rats (RLI) or male Syrian hamsters (HLI).
Test concentrations with justification for top dose:
Main test (2 experiments):
Trial 1: 0 (solvent control), 3, 10, 33, 100, 333 and 666 μg/plate with S9 mix, and 0 (solvent control), 3, 10, 33, 100 and 333 μg/plate without S9 mix.
Trial 2: 0 (solvent control), 3, 10, 33, 100 and 333 μg/plate with and without S9 mix.
The final dose level selection was based on the results of a preliminary range-finding study conducted with TA100 in the presence and absence of S9.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle: insoluble in water
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 4-nitro-o-phenylenediamine: -S9: TA98; 2-aminoanthracene: +S9: all strains.
Details on test system and experimental conditions:
METHOD OF APPLICATION: Preincubation assay

DURATION
- Preincubation period: 20 min at 37ºC
- Exposure duration: 48 hours at 37ºC

SELECTION AGENT (mutation assays): the lack of amino-acid (Histidine) in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 2 independent tests with 3 plates/dose/strain.

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity was evidenced by one or more of the following phenomena: appearance of his- pinpoint colonies, reduced numbers of revertant colonies per plate, or thinning or absence of the bacterial lawn.




Evaluation criteria:
A positive response is defined as a reproducible, dose-related increase in histidine-independent (revertant) colonies in any one strain/activation combination. An equivocal response is defined as an increase in revertants that is not dose related, is not reproducible, or is not of sufficient magnitude to support a determination of mutagenicity. A negative response is obtained when no increase in revertant colonies is observed following chemical treatment. There is no minimum percentage or fold increase required for a chemical to be judged positive or weakly positive.
Statistics:
No statistical methods were used.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Slight toxicity at 333 μg/plate (-S9) and 666 μg/plate (+S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Slight toxicity at 333 μg/plate (-S9 and +S9 HLI, trial1) and toxic at 666 μg/plate (+S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Slight toxicity at 333 μg/plate and 666 μg/plate (+S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Slight toxicity at 333 μg/plate (-S9) and 666 μg/plate (+S9 RLI) and toxic at 666 μg/plate (+S9 HLI)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
A preliminary range-finding study was conducted with TA100 in the presence and absence of S9.

Any other information on results incl. tables

Table 1: Mutagenicity of Methyleugenol in Salmonella typhimurium (a)

Strain

Dose [µg/plate]

Revertants/plate b

-S9

+10% hamster S9

+10% rat S9

Trial 1

Trial 2

Trial 1

Trial 2

Trial 1

Trial 2

TA100

0

120 ± 3.2

90 ± 6.4

106 ± 4.4

103 ± 8.7

111 ± 7.8

98 ± 8.1

3

101 ± 0.0

86 ± 3.5

 

90 ± 8.0

 

95 ± 5.3

10

100 ± 6.4

93 ± 4.0

106 ± 4.6

89 ± 6.1

93 ± 3.3

94 ± 2.7

33

114 ± 8.3

93 ± 10.7

109 ± 4.6

90 ± 6.8

109 ± 4.4

92 ± 4.3

100

105 ± 11.6

96 ± 2.7

116 ± 7.0

80 ± 14.4

110 ± 0.7

91 ± 7.6

333

29 ± 8.2c

16 ± 13.1c

99 ± 4.2

78 ± 1.0

95 ± 9.4

97 ± 2.6

666

 

 

38 ± 38.0c

 

0 ± 0.0c

 

Trial summary

Negative

Negative

Negative

Negative

Negative

Negative

Positive control d

358 ± 7.0

388 ± 4.3

1469 ± 61.2

1111 ± 49.2

534 ± 46.0

351 ± 22.9

TA1535

0

24 ± 2.6

20 ± 3.5

10 ± 2.4

12 ± 2.1

10 ± 2.2

9 ± 0.6

3

37 ± 0.6

20 ± 2.3

 

8 ± 0.9

 

6 ± 0.0

10

33 ± 2.0

21 ± 2.3

13 ± 3.5

8 ± 2.3

8 ± 0.9

7 ± 0.3

33

37 ± 7.5

22 ± 3.3

14 ± 1.5

9 ± 2.8

9 ± 0.0

9 ± 2.6

100

32 ± 3.5

26 ± 2.7

11 ± 2.2

10 ± 3.7

6 ± 1.5

7 ± 1.0

333

5 ± 5.0c

2 ± 0.7c

10 ± 2.1

9 ± 2.3

4 ± 0.3

8 ± 0.9

666

 

 

4 ± 2.6c

 

0 ± 0.0c

 

Trial summary

Negative

Negative

Negative

Negative

Negative

Negative

Positive control

375 ± 15.5

410 ± 15.2

381 ± 7.9

369 ± 20.8

146 ± 2.8

168 ± 21.2

TA1537

0

5 ± 0.6

5 ± 0.3

6 ± 1.0

5 ± 0.3

7 ± 0.7

6 ± 0.9

3

6 ± 1.5

3 ± 0.9

 

9 ± 1.5

 

8 ± 2.1

10

4 ± 1.2

3 ± 0.9

5 ± 1.2

6 ± 0.9

5 ± 0.9

4 ± 1.0

33

5 ± 0.3

4 ± 1.2

5 ± 1.5

5 ± 1.2

4 ± 0.3

9 ± 1.5

100

4 ± 1.5

4 ± 0.6

6 ± 1.5

5 ± 1.0

7 ± 0.0

7 ± 1.2

333

0 ± 0.0c

3 ± 0.03c

3 ± 1.2

4 ± 1.3

6 ± 0.3

5 ± 2.2

666

 

 

Toxic

 

0 ± 0.0c

 

Trial summary

Negative

Negative

Negative

Negative

Negative

Negative

Positive control

184 ± 4.7

521 ± 48.1

424 ± 75.9

426 ± 15.5

157 ± 8.7

124 ± 11.9

TA98

0

15 ± 0.6

16 ± 1.7

25 ± 2.2

31 ± 3.7

17 ± 3.6

20 ± 4.1

3

15 ± 0.7

13 ± 2.2

 

31 ± 4.0

 

27 ± 0.9

10

15 ± 1.0

14 ± 0.9

27 ± 3.5

28 ± 2.3

24 ± 2.7

23 ± 2.6

33

15 ± 0.9

13 ± 1.8

31 ± 5.8

26 ± 1.2

29 ± 2.0

20 ± 2.3

100

14 ± 3.5

13 ± 0.9

28 ± 0.7

29 ± 6.0

20 ± 3.5

29 ± 5.5

333

0 ± 0.0c

3 ± 3.0c

15 ± 7.7c

21 ± 2.7

29 ± 5.0

19 ± 0.3

666

 

 

Toxic

 

Toxic

 

Trial summary

Negative

Negative

Negative

Negative

Negative

Negative

Positive control

626 ± 20.6

412 ± 8.2

1274 ± 85.7

1362 ± 55.5

473 ± 34.3

444 ± 76.4

a Study performed at SRI International. The detailed protocol is presented by Mortelmans et al. (1986). 0 μg/plate dose was the solvent control.

b Revertants are presented as mean ± the standard error from three plates.

c Slight toxicity

d The positive controls in the absence of metabolic activation were sodium azide (TA100 and TA1535), 4-nitro-o-phenylenediamine (TA98), and 9-aminoacridine (TA1537). The positive control for metabolic activation with all strains was 2-aminoanthracene.

Applicant's summary and conclusion

Conclusions:
Methyl eugenol was not mutagenic with Salmonella typhimurium strains TA100, TA98, TA1535 and TA1537 with and without metabolic activation.

Executive summary:

In a bacterial reverse mutation assay following a method similar to OECD guideline 471, the test item methyl eugenol diluted in Dimethylsulfoxide (DMSO) was tested with Salmonella typhimurium strains TA100, TA98, TA1535 and TA1537 with and without metabolic activation (S9) using the preincubation method. Based on results of a preliminary assay, for the main test two independent experiments and 3 replicates of each were conducted with doses of 0 (solvent control), 3, 10, 33, 100 and 333 μg/plate with and without S9 mix. Also a dose of 666 μg/plate with S9 mix was used in the first experiment. Concurrent solvent and positive controls were included in every experiment and their responses were considered valid. The test item did not show mutagenic activity in any of the bacterial strains tested.